sign in sign up
<<
Home , Rickettsia, Rickettsia japonica

INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1992, p. 303-305 Vol. 42, No. 2 0020-77 13/92/020303 -03 $02. 0010 Copyright 0 1992, International Union of Microbiological Societies

Rickettsia japonica sp, nov, the Etiological Agent of Group in Japan

TAKAHIRO UCHIDA,l* TSUNEO UCHIYAMA,' KEIKO KUMAN0,l AND DAVID H. WALKER2 Department of Virology, School of Medicine, University of Tokushima, Tokushima 770, Japan' and Department of Pathology, University of Texas Medical Branch, Galveston, Texas 775502

We propose the name japonica sp. nov. (with type strain YH [= ATCC VR-13631) for a serologically specific of spotted fever group rickettsiae that are pathogenic for humans (J. Infect. Dis. 159:1122-1126, 1989; J. Clin. Microbiol. 28:1177-1180, 1990). The biologic and genomic characteristics of the organism (G+C content, 31.2 +- 0.7 mol%) are essentially the same as those of other pathogenic spotted fever group rickettsiae, although the R. japonica isolates cause a persistent infection in Vero cells for many subcultures.

In this paper we formally describe (23, YHT) that reacts with all of the strains of SFG rickettsiae 25), which has been identified as the causative agent of a tested but not with Wilmington (= ATCC human disease. The first isolate, strain YHT (T = type VR144). Polyclonal antibodies produced in mice against R. strain), was isolated in 1985 from the blood of a patient with japonica react with R. typhi only at low titers. On the other febrile exanthematous illness in Japan by using a hand, sera from some patients infected with R. japonica culture technique (20). Five strains of the causative agent, react with R. typhi at high dilutions (22). including strain YHT, have been isolated from patients that Growth of R. japonica occurs in Vero cells without were serodiagnosed as having a spotted fever group (SFG) cytopathic effects, and a carrier state that persists for 20 rickettsiosis (22). subcultures of infected cells develops. Carrier Vero cells Justification for a new species. The justification for estab- divide until a monolayer is formed after subculturing in lishing a new species for this organism is based on the minimal essential medium containing 10% fetal calf serum current standard method for comparing rickettsial taxo- and no in a 5% C02-air incubator at 34°C. After nomic types by serologic analysis (23). Experiments involv- cocultivation with uninfected Vero cells, all cells enter the ing reciprocal cross-reactions of mouse polyclonal antibod- carrier state. The organisms in carrier cells are viable after ies to strains of the new species and other species of SFG storage at -80°C or in liquid nitrogen when the carrier cells rickettsiae produced by the standard method were carried are stored in minimal essential medium containing 5% di- out to calculate the specificity differences. By using this methyl sulfoxide, 20% fetal calf serum, and no antibiotics. R. approach we demonstrated that all five strains belong to a japonica differs from other SFG rickettsiae with respect to single species that is distinct from all of the previously growth in Vero cells; the strains which we tested have been described SFG rickettsiae that are known to be pathogenic described previously (23). Cutlack, Rick- for humans (23). In addition, none of the isolates reacted ettsia conorii Malish 7, R (= ATCC with mouse monoclonal antibodies that are species specific VR891), and Thai rickettsia TT-118 produce for other pathogenic SFG rickettsiae (23). Furthermore, the cytopathic effects 3 to 6 days after inoculation. Although results of Western immunoblotting revealed different elec- trophoretic mobilities and antigenic reactivities for the major Kaplan (= ATCC VR148) and Rickettsia immunodominant high-molecular-weight surface polypep- sibirica 232 cause few cytopathic effects, the infected cells tides of the Japanese isolates and standard pathogenic SFG do not grow continuously after subculturing. The growth rickettsial strains (23). Species-specific monoclonal antibod- properties are the same in Vero C1008 (= ATCC CRL1586) ies to R. japonica reacted only with strains of R. japonica, cells. Chicken fibroblast primary culture, BHK supporting the conclusion that R. japonica is a new species 21/13, and L929 cells allow R. japonica to grow with of SFG rickettsiae (25). cytopathic effects. Replication in Vero and BHK 21/13 cells Description of Rickettsiajaponica sp. nov. Rickettsia japon- occurs primarily in the cytoplasm and rarely in nuclei. Figure 1 shows ultrathin sections of heavily infected Vero ica (ja. PO' ni. ca. N.L. adj. japonica, pertaining to Japan, the country in which the organism was first isolated). In C1008 cells, which were examined with a Hitachi model smears from cell cultures, the intracellular organisms are HU-12 electron microscope as described previously (24). visualized by Gimenez or by indirect immunofluo- The plaque morphology produced by R. japonica is dif- rescence with immune sera (20). Gram-negative rods that are ferent from that produced by most other SFG rickettsiae. 0.4 to 0.5 by 0.8 to 1.5 km. The organisms possess an outer Infected Vero cells centrifuged at 250 x g for 5 min were slime layer and a trilaminar cell wall with thin outer and thick diluted in sucrose-phosphate-glutamate buffer (1) and plated inner leaflets (24). Obligate . The organ- onto Vero cell monolayers and then overlaid with Leibovitz isms carry species-specific epitopes on the surface (25). The medium L-15 containing 0.5% methylcellulose and 5% fetal organisms also possess an SFG-common antigen that is calf serum. At intervals during incubation at 34°C in an air detectable by monoclonal antibody 3Y8-B3 (to R. japonica incubator, the monolayers were simultaneously fixed and stained with a 10% formalin solution containing 0.5% crystal violet and washed with water to identify plaques (infectious centers). After 9 to 11 days of incubation, R. japonica * Corresponding author. formed plaques with a targetlike appearance (a dye-stained

303 304 NOTES INT. J. SYST. BACTERIOL.

TABLE 1. T, values and G+C contents of DNAs"

G+C content (mol %) T, ("C) determined: determinedh:

R. japonica YH 82.1 f 0.3 31.2 f 0.7 R. rickettsii R 82.3 2 0.2 82.4 5 0.2 31.7 & 0.5 32.0 f 0.5 E. coli B 90.5 f 0.2 90.5 51.7 f 0.5 51.7 DNAs were extracted from Percoll density gradient-purified rickettsiae by treatments with lysozyme (3 mg/ml at 37°C for 30 min) and proteinase K (30 p&ml in 1% sodium dodecyl sulfate at 50°C for 3 h), followed by a phenol-chloroform treatment (12) that included DNase-free RNase A treat- ment (0.1 mg/ml at 37°C for 2 h). Calculated by using the formula of Marmur and Doty (9). c' Means & standard deviations for three to five determinations. Data from references 9 and 17. Ultrapure genomic DNA (Sigma Chemical Co.) was used as a standard.

targetlike plaques; the one exception was a clear-plaque variant that was isolated from a guinea pig which was inoculated with lo6 carrier cells. Specific--free strain C3H/HeSlc mice and conventional strain ddY mice (males that were 8 to 12 weeks old) developed inapparent infections after intraperitoneal inoculation; C3H/He mouse strains have been reported to be susceptible to R. conorii (3). The mice produced antibodies (titers, 1:20 to 1:160) on day 7, and the titers increased to 1:320 to 1:2,560 on day 21 after inoculation with lo2 to lo3 carrier cells. Chicken died 5 to 7 days after yolk sac inoculation with organisms propagated in chicken embryo fibroblasts ( lo3 infected cells) when they were incubated at 33°C; the maximum yield FIG. 1. (A) Rickettsiae in the cytoplasm of heavily infected Vero occurred on day 2 after the death of the chicken embryos. C1008 cells. (B) Rickettsiae in a nucleus (N). The arrowhead R. juponicu exhibited no hemolytic activity when it was indicates the nucleolus. Bars = 1.5 ym. assayed with 0.26 mg of protein by using the method of Snyder et al. (14); the organisms which we used were propagated in BHK 21/13 cells and banded at a density of inner spot surrounded by a clear zone). The diameter of the 1.080 g/cm3 in a Percoll gradient. A control reaction mixture targetlike plaques increased to 2.0 mm on day 13. Other SFG containing the same quantity of R. typhi protein resulted in rickettsiae produced clear plaques on day 6; R. rickettsii hemolysis, giving an A,,, of 0.240. produced plaques with diameters of 1.6 and 2.0 mm on days The buoyant density of R. japonica DNA was estimated to 8 and 11, respectively, while other rickettsiae formed smaller be 1.692 g/cm3. Rickettsia1 DNA extracted from Percoll plaques. R. akari produced very tiny plaques. When mono- density gradient-purified organisms by using the standard layers were overlaid with 0.8% agarose and stained with phenol-chloroform method (12) was centrifuged with CsCl in neutral red as described previously (2), no plaques were 10 mM Tris HCI (pH 8.0) at 290,000 x g for 7 h at 20°C in a formed by R. japonica even after 13 days, while clear type RP55VF2 vertical rotor in a Hitachi model CP56G plaques with a diameter of 2.5 mm were produced by R. Himac preparative ultracentrifuge, resulting in a single peak rickettsii after 8 days of incubation. A plaque type variant of DNA in a CsCl gradient. The CsCl density of the peak isolated from a guinea pig after inoculation with R. japonica fraction, obtained from the refractory index (5),was deter- (see below) formed a clear plaque. About 0.01 to 0.001% of mined on the basis of a value of 1.710 g/cm' for the density the carrier cells with strain YHT produced clear plaques on of the reference DNA from B (13). Both Vero cell monolayers. strain YHT and clear-plaque variant DNAs banded at the The organisms are pathogenic for guinea pigs. Specific- same density. pathogen-free male guinea pigs (strain Hartley, 13 weeks The thermal denaturation temperature (T,) of DNA from old) were obtained from the SLC Farm, Shizuoka, Japan, R. japonica was 82.1 5 0.3"C (n = 5). A Hitachi model and were inoculated intraperitoneally with 10 to lo6 carrier U-3210 spectrophotometer equipped with a thermoelectric cells that had been subcultured 9 to 11 times and contained cell holder connected to a model SPR-10 temperature con- an average of 50 rickettsia1 particles per cell, which were troller and a digital thermometer was used to measure the T, counted after smears were stained by using the immunoflu- as described by Marmur and Doty (9). In this study we used orescence technique. The rectal temperatures abruptly in- clear-plaque variant DNA. As Table 1 shows, reference R. creased to more than 40°C; this reaction was accompanied rickettsii R DNA, which was extracted and purified as by scrota1 swelling 2 to 3 days after inoculation, even if a described above, had a T, identical to the value reported small dose (10 carrier cells) was inoculated. The fever previously (17). An E. coli B standard DNA also had the T, continued for 2 to 3 days and was followed by defervescence reported previously (9). to around 38°C. Most of the rickettsiae recovered by tissue The guanine-plus-cytosine (G+C) content of R. japonica culturing from the blood during the febrile stage produced DNA calculated from the T, by using the previously de- VOL.42, 1992 NOTES 305 scribed formula (9) was 31.2 2 0.7 mol%. This value is Suto, Y. Tsuboi, A. Oya, H. Koyama, T. Uchiyama, and T. identical to the values obtained for other SFG rickettsiae Uchida. 1985. The first report of rickettsial infections of spotted (17). The G+C value calculated from the estimated buoyant fever group in Japan; three clinical cases. Kansenshogaku density by using the equation described by Schildkraut et al. Zasshi 591165-1172. (In Japanese.) (13) was 32.6 mol%. 9. Marmur, J., and P. Doty. 1962. Determination of the base Description of the illness. The illness caused by R. japonica composition of deoxyribonucleic acid from its thermal denatur- is found mainly in the southwestern part of Japan (4,6-8, 15, ation temperature. J. Mol. Biol. 5109-118. 10. Oka, N., Y. Kato, S. Dekio, T. Nishio, A. Itagaki, T. Uchiyama, 16, 18, 19, 21, 26); a few cases have been reported in the and T. Uchida. 1990. A case report of spotted fever group west-northwest portion of Honshu (10). R. japonica causes rickettsiosis first encountered in Shimane Prefecture, Japan. an illness (Oriental spotted fever) that is similar to bouton- Kansenshogaku Zasshi 64:136-142. (In Japanese.) neuse fever. A similar agent has been isolated in another 11. Okada, T., Y. Tange, and Y. Kobayashi. 1990. Causative agent laboratory (11) and remains to be identified as R. japonica. of spotted fever group rickettsiosis in Japan. Infect. Immun. The vector has not been determined. The full geographic 58:887-892. distribution of the organisms remains to be established. 12. Sambrook, J., E. F. Fritsch, and T. Maniatis (ed.). 1989. Type strain. Type strain YH (= ATCC VR-1363) of R. Molecular cloning. A laboratory manual, 2nd ed. Cold Spring japonica is the first strain of R. japonica that was isolated Harbor Laboratory Press, Cold Spring Harbor, N.Y. and has been distributed to the National Institute of Health, 13. Schildkraut, C. L., J. Marmur, and P. Doty. 1962. Determina- Tokyo, Japan, and to prefectural public health laboratories tion of the base composition of deoxyribonucleic acid from its in Japan. All of the strains have the same properties. buoyant density in CsCl. J. Mol. Biol. 4430443. 14. Snyder, J. C., M. R. Bovarnick, J. C. Miller, and R. S.-M. We are grateful to Charles L. Wisseman, Jr. (University of Chang. 1954. Observations on the hemolytic properties of Maryland) for his advice and to Akiyoshi Kawamura, Jr. (University typhus rickettsiae. J. Bacteriol. 67:724-730. of Tokyo) and Hiroshi Tanaka (University of Tokyo) for their 15. Suto, T. 1985. Evidence of spotted fever rickettsial infection in encouragement. We also acknowledge Takashi Kobunai (Biological Japan as demonstrated by the indirect immunoperoxidase test. Research Laboratory, Taiho Pharmaceutical Co., Tokushima, Ja- Microbiol. Immunol. 29:1243-1246. pan) for determining the TIP,of the DNA. 16. Tachibana, N., E. Shishime, A. Okayama, J. Ishizaki, K. Murai, This work was supported in part by grant 63044102 for joint S. Shioiri, K. Tsuda, and T. Oshikawa. 1987. Two cases of research from the Monbusho International Scientific Research Pro- spotted fever rickettsiosis in Kyushu. Kansenshogaku Zasshi gram of the Ministry of Education, Science and Culture of Japan, by 61:1166-1172. (In Japanese.) a grant from the Ohyama Health Foundation, Tokyo, Japan, and by 17. Tyeryar, F. J., Jr., E. Weiss, D. B. Millar, F. M. Bozeman, and grant AI-22224 from the National Institute of Allergy and Infectious R. A. Ormsbee. 1973. DNA base composition of rickettsiae. Diseases. Science 180:415-417. 18. Uchida, T., F. Mahara, Y. Tsuboi, and A. Oya. 1985. Spotted REFERENCES fever group rickettsiosis in Japan. Jpn. J. Med. Sci. Biol. 1. Bovarnick, M. R., J. C. Miller, and J. C. Snyder. 1950. The 38: 151-153. influence of certain salts, amino acids, sugars, and proteins on 19. Uchida, T., F. Tashiro, T. Funato, and Y. Kitamura. 1986. the stability of rickettsiae. J. Bacteriol. 59509-522. Immunofluorescence test with Rickettsia montana for serologic 2. Cory, J., C. E. Yunker, R. A. Ormsbee, M. Peacock, H. Meibos, diagnosis of rickettsial infection of the spotted fever group in and G. Tallent. 1974. Plaque assay of rickettsiae in a mammalian Shikoku, Japan. Microbiol. Immunol. 30:1061-1066. cell line. Appl. Microbiol. 27:1157-1161. 20. Uchida, T., F. Tashiro, T. Funato, and Y. Kitamura. 1986. 3. Eisemann, C. S., M. J. Nypaver, and J. V. Osterman. 1984. Isolation of a spotted fever group rickettsia from a patient with Susceptibility of inbred mice to rickettsiae of the spotted fever febrile exanthematous illness in Shikoku, Japan. Microbiol. group. Infect. Immun. 43:143-148. Immunol. 30: 1323-1326. 4. Funato, T., Y. Kitamura, A. Kawamura, and T. Uchida. 1988. 21. Uchida, T., Y. Tsuboi, A. Oya, T. Funato, and Y. Kitamura. Rickettsiosis of spotted fever group encountered in Muroto area 1986. Serologic studies of spotted fever group rickettsiosis of Shikoku, Japan: clinical and epidemiological features of 23 occurring in Muroto-City, Kochi Prefecture. Kansenshogaku cases. Kansenshogaku Zasshi 62:783-791. (In Japanese.) Zasshi 60141-144. (In Japanese.) 5. If€t, J. B., D. H. Voet, and J. Vinograd. 1961. The determination 22. Uchida, T., T. Uchiyama, and A. H. Koyama. 1988. Isolation of of density distributions and density gradients in binary solutions spotted fever group rickettsiae from humans in Japan. J. Infect. at equilibrium in the ultracentrifuge. J. Phys. Chem. 651138- Dis. 158:664-665. 1145. 23. Uchida, T., X. J. Yu, T. Uchiyama, and D. H. Walker. 1989. 6. Iwamoto, K., F. Nishimura, Y. Yoshino, J. Mihara, T. Okabe, H. Identification of a unique spotted fever group rickettsia from Kameda, and T. Kubagawa. 1988. A case of spotted fever with humans in Japan. J. Infect. Dis. 159:1122-1126. central nervous system involvement. Kansenshogaku Zasshi 24. Uchiyama, T., and T. Uchida. 1988. Ultrastructural study on 62:1192-1196. (In Japanese.) Japanese isolates of spotted fever group rickettsiae. Microbiol. 7. Kaiho, I., M. Tokieda, M. Ohtawara, T. Uchiyama, and T. Immunol. 32:1163-1166. Uchida. 1988. Occurrence of rickettsiosis of spotted fever group 25. Uchiyama, T., T. Uchida, and D. H. Walker. 1990. Species- in Chiba Prefecture of Japan. Jpn. J. Med. Sci. Biol. 41:69-71. specific monoclonal antibodies to Rickettsia japonica, a newly 7a.Kodama, K., T. Matsuo, M. Nakauchi, Y. Kobayashi, Y. Tange, identified spotted fever group rickettsia. J. Clin. Microbiol. and T. Okada. 1990. Spotted fever group rickettsiosis on Awaji 28: 1177-1180. Island, Japan. Kansenshogaku Zasshi 64504-509. (In Japa- 26. Yamamoto, S., N. Kawabata, T. Uchiyama, and T. Uchida. 1987. nese.) Evidence for infection caused by spotted fever group rickettsia 8. Mahara, F., K. Koga, S. Sawada, T. Taniguchi, F. Shigemi, T. in Kyushu, Japan. Jpn. J. Med. Sci. Biol. 40:75-78.